1. Field of the Invention
The invention relates to a method for the microsurgical, cellular repair of mutated sections of an RNA and for the specific destruction of tumor cells with the aid of an incorporated RNA capable of trans-splicing, using naturally occurring splicing components in the cell, and to a screening method for the detection of naturally trans-spliced cellular RNA.
2. Prior Art
In mammal cells, splicing processes can take place within a single RNA sequence, i.e. cis-splicing, and between separate RNA sequences, i.e. trans-splicing. Trans-splicing means that bonds within an RNA molecule are cleaved and new bonds with other RNA molecules are formed, thus producing a new RNA molecule. Such RNA trans-splicing processes therefore modify the genetic information and may give rise to modified RNA molecules or pathogenic mutations inducing tumors, for example. Providing a method of identifying trans-spliced RNA would therefore be of great importance. However, trans-splicing processes can also be used in a well-aimed fashion to repair a mutated gene or e.g. specifically destroy tumor cells. For this reason, the present inventors seek protection by patents for a basic process and for a product by means of which, depending on the case of application, trans-spliced RNA for therapeutic purposes is produced, or possibly pathogenic, cellularly trans-spliced RNA can be diagnosed.
These uses are based on the procedural basic principle of producing an artificial pre-mRNA by means of a DNA that is introduced into living cells, which pre-mRNA differs substantially in its structure from all natural cellular pre-mRNA molecules: namely, said artificial pre-mRNA comprises only 1 or 2 splice sites at maximum, corresponding to an exon flanked at both of its ends by one or two “intron moieties”, herein defined as outrons. Depending on the case of application, this exon then is used to replace/repair a genetically defective exon of a natural cellular RNA by means of a replacement procedure (application principle 1), or in tumor cells, for example, after coupling to an exon from a tumor-specific RNA, causing formation of an mRNA encoding a protein which directly or indirectly induces cell breakdown of the tumor cell (application principle 2), or the exon is part of an RNA probe by means of which cellular pre-mRNAs capable of trans-splicing are identified at first, which optionally interact to form diagnosable new hybrid mRNA molecules in the cell (case of application 3), which in turn trigger possibly pathogenic processes.
In addition, the RNA which is used in each of the three cases and correspondingly includes one or two splice sites must meet specific requirements in each case to ensure sufficient functional efficiency: in order to exhibit high potency in intermolecular trans-splicing, the splice sites are required to include sequences allowing optimum binding of splicing (helper) proteins to these RNA sequences. When using this RNA for therapeutic purposes (case of application 1 or 2), specific binding between the incorporated RNA capable of trans-splicing and the cellular target RNA is required in addition, which is achieved by means of an artificially generated antisense structure on the incorporated RNA, which undergoes specific pairing with the cellular target RNA in this region. The ionic-chemical bond (via hydrogen ion bridges) thus formed between the incorporated RNA and the cellular target RNA substantially increases the trans-splicing efficiency.
Referring to the application principle 1, namely, using the incorporated RNA as repair RNA, it should be noted that a large number of diseases, such as Alzheimer, Parkinson, diabetes, hemophilia B, hereditary hypertension etc., are triggered by congenital or later-acquired singular gene defects or mutations. At present, the above diseases of monogenetic cause are generally treated using medicaments in such a way that these medicaments have a purely symptom-related physiological effect, without concerning the actual causes of the clinical picture.
If the genetic defect becomes manifest only in particular cell types or in organs of specific function, substitution and replacement of such cells or organs via trans-plantation is possible as an alternative. However, due to the unresolved problems of immune rejection of heterotransplants and the risk of virus transfer, the use of these techniques is limited.
Up to now, there are only a few cases of an at least causal treatment by external supply of a protein that is missing or non-functional as a result of a genetic defect. Examples are the daily injection of insulin in diabetics or of specific blood coagulation factors in hemophilia.
In contrast to symptomatic or causal treatment, a proper cure of genetic diseases is, in principle, only possible by restoring the function of the defective or mutated gene in the cells. On a molecular level, microsurgical gene repair by specific replacement of the defective gene components in the cell is not possible according to the present state of the art, and for this reason, the defective gene is replaced in its function by introducing a homologous, intact gene according to established methods of gene therapy.
Due to various technical problems, such as limited load capacity of viral gene shuttles, it is not the actual gene consisting of many exon and intron regions, some of them being 100 kb or more in size, that is used in gene replacement. Rather, the transgene introduced into the cells is a cDNA of the gene, which is by up to 95% shorter and merely consists of the protein-encoding exon portions, and is obtained upon reverse transcription of the mRNA. At its N terminus this cDNA has a suitable promoter and at the C terminus a recognition site for RNA polyadenylation, e.g. from SV40, coupled thereto by genetic engineering. Above all, polyadenylation is responsible for the protection of the mRNA against cytoplasmatic RNases and thus for the stability thereof. Such gene-therapeutic cDNA constructs allow constant and high RNA expression. However, replacement of a defective gene, with its complex structure, especially its intron portions and regulatory sequences, with a compressed cDNA homologue involves serious disadvantages:
a) A very large number (or percentage) of cellular primary gene transcripts—the pre-mRNAs—not only encode a single mRNA or a single protein, but are composed into most various mRNAs and proteins via alternative splicing processes. In cellular replacement of a gene on a cDNA rather than a DNA level, the mechanism of RNA maturing via splicing does no longer apply. For each required version of these mRNAs, another cDNA therefore must be introduced into the cell by gene therapy.
b) Furthermore, the selection of exons and thus the type of proteins formed in alternative RNA splicing in the cell is controlled by highly complex mechanisms which in turn are controlled by various external factors. Helper proteins, in particular, bind to regulatory RNA sequences, thereby influencing the selection of exons in alternative splicing processes and thus the mutual molar ratio of all sorts of possible, alternative splicing products. Moreover, the ratio of alternative splicing products with respect to each other can be subject to a complex process control in time, such as the various splicing forms in immunoglobulins (IgM, IgG etc.). Frequently, however, it is precisely the introns which include the regulatory sequences for alternative splicing processes. Thus, even in those cases where all sorts of possible alternative mRNA forms were available as a result of various cDNA constructs introduced (see a), physiological—and frequently also time-controlled—quantitative regulation of these mRNAs with respect to each other is absent, because the regulatory intron sequences are not included in such cDNAs. Replacement of alternatively spliceable defective genes by means of conventional methods using a cDNA lacking these intron structures is therefore disadvantageous.
c) Another essential aspect is that regulatory sequences for transcription control, such as enhancers etc., sometimes are situated at a great N-terminal distance from the promoter, or even in intron regions. However, artificial cDNA gene constructs no longer have this natural background, i.e., the intron regions are absent (see above) as are the regulatory sequences which are often far away from the promoter on the non-transcribed DNA. Thus, even when using the authentic promoter, cDNA constructs no longer exhibit the cellular fine regulation of transcription and therefore, non-physiological over- or underexpression of proteins may occur, which in turn can do damage to cells.
Therefore, due to major DNA portions that are missing (introns and other regions are absent), an artificially introduced, compressed replacement cDNA provided with a promoter and a new polyadenylation site can never have the complexity of the homologous natural, genetically defective gene and consequently cannot be a physiologically adequate substitute.
Where large genetically defective mRNAs or proteins (molecular weight: 150-200 kDa) are to be replaced, a transgene size of 5 kb and longer is required even when using a compressed cDNA. Such a size may result in substantial restrictions when using particular gene shuttles (e.g. adeno-associated virus (AAV)), and especially in nonvirus-mediated, direct gene transfer.